On the Lachrymatory Factor in Onion (Allium cepa) Vapours and Its Precursor.

Abstract

cf. Virtanen and Spare, CA 56, 10585g. A lachrymatory test was devised in order to follow the isolation of the precursor (I) of the lachrymatory factor (II) in Allium cepa. A I-free enzyme-contg. powder was prepd. by homogenizing onions in distd. H2O, leaving for 2 hrs. with occasional stirring, and lyophilizing the sludge. Approx. 5 mg. of this powder was added to 4-6 drops of the aq. soln. (pH 6-7) to be tested in a weighing bottle. The suspension was stirred for 10 sec. and the bottle held tightly to the eye. The lachrymatory effect was observed after 15-45 sec. Using this test, I was isolated from onions. Whole onions were frozen to -80° with solid CO2, crushed, and homogenized in 70% EtOH at -80° to inactivate the enzymes. The suspension was filtered and the extn. with 70% EtOH repeated at room temp. The pooled clear exts. were passed through an Amberlite IR-120 (H+) column and eluted with N NH4OH. The effluent was continuously and rapidly taken to dryness in vacuo with the exclusion of O to minimize conversion to cycloalliin. The residue was dissolved in 0.5N AcOH and chromatographed on a Dowex 1-X8 column (acetate form). Fractions contg. neutral amino acids were dissolved in 0.5N pyridine buffer (pH 2.7 with HCOOH) and fractionated on a Dowex 50-X8 column. I, cycloalliin, and S-methylcysteine were the 1st major amino acids to be eluted. I-contg. fractions were purified on a cellulose column using BuOH-AcOH-H2O (63:10:27), and I was pptd. twice from aq. Me2CO and aq. EtOH. Pure I (129 mg.), a white cryst. solid (decomp. 146-148°; [α]25D approx. + 74°) and 200 mg. of less-pure I were obtained from 5 kg. of fresh onions. Elementary analysis, enzymic cleavage, hydrolysis, and infrared spectroscopy showed I to be (+)-S-(1-propenyl)cysteine sulfoxide. It was stable in neutral soln. or in 2N AcOH, but in mild alk. soln., it was converted to cycloalliin. Acid hydrolysis split I into cysteine, propionaldehyde, and S. Enzymic cleavage converted I into II, pyruvic acid, NH3, and some propionaldehyde. No amino acids were detected during the enzymic splitting of I, nor was dipropenylthiosulfenate formed. Mass spectral studies revealed no mass higher than 90 among the products. As II was very unstable, proof of its structure was largely obtained from the mass-spectral studies. It appears to be propenylsulfenic acid, the first aliphatic sulfenic acid to be identified: CH3CH:CHSHO. II spontaneously changes into propenyl alc. by loss of S, and this rearranges into propionaldehyde. Part of the latter compd. is further condensed into 2-methyl-2-pentenal. Gel-filtration on Sephadex was the best method of purifying the I-splitting enzyme, and evidence was obtained that there are 2 I-splitting enzymes in onion. [on SciFinder(R)]